| ObjectiveDistraction Osteogenesis(DO)is an important technique commonly used in oral and maxillofacial surgery.It has a fast rate of osteogenesis and high controllability,and it is one of the hotspots of research at home and abroad.In the previous research,our group found that there is an abundant blood vessel formation process in the distraction zone,along with the osteogenesis.Endothelial progenitor cells(EPCs)will directly participate in the neovascularization during distraction osteogenesis.And Non-coding RNA plays a key role in regulating neovascularization in a variety of physiological or pathological processes.The m RNA in the previous high-throughput sequencing results of the research group is analyzed,which shows that KLF4 plays an important role in neovascularization.We aim to explore the non-coding RNA that regulates neovascularization of endothelial progenitor cell,that is,to explore the mechanism of mi RNA7 and lnc RNA193740 targeting KLF4 to regulate endothelial progenitor cell neovascularization during distraction osteogenesis.Methods1.Filtrate the m RNA and mi RNA which is associated with neovascularization,and conduct preliminary verification: Through the analysis of the early high-throughput sequencing results,it was found that compared with bone fracture(BF)tissues,the DO tissues had significantly differently expressed m RNA.Through bioinformatics analysis,we’d like to determine the significant differential expression and angiogenic function related m RNA.The upstream mi RNA targeting m RNA is screened by bioinformatics analysis.The relative expression levels of mi RNA and m RNA in the tissues were detected by q RT-PCR.The targeted regulation relationship between mi RNA and m RNA was validated via luciferase experiment.2.In vitro experimental study of mi RNA7 targeting KLF4 to regulate EPCs neovascularization:(1)Isolation,culture and identification of neovascularization ability of endothelial progenitor cells: the bone marrow of healthy puppies was taken,and EPCs were isolated by density gradient centrifugation and cultured.Identification:1)Observe the morphological characteristics of EPCs by inverted microscope;2)identify EPCs by double fluorescence labeling;3)detect EPCs surface markers by immunofluorescence;4)detect the migration ability and tube formation of EPCs in vitro by cell migration experiment and cell tube formation experiment.(2)Overexpression and silence of mi RNA7 in EPCs were constructed by Lentiviruses.The effects of mi RNA7 on the neovascularization function of EPCs were detected by CCK8,cell migration experiments,and cell tube formation experiments.The expression levels of KLF4,VEGFA,and b FGF in the cells were detected by q RT-PCR,and WB was used to detect the protein levels of KLF4,VEGFA,b FGF.3.Preliminary exploration of lnc RNA193740 in regulation of KLF4: Based on the theory of ce RNA,combined with bioinformatics analysis,high-throughput sequencing results were screened to obtain lnc RNA193740 that may be involved in KLF4 regulation.Construct a Lentiviruses to overexpress lnc RNA193740 in EPCs,and detect the effect of lnc RNA193740 on the neovascularization function of EPCs by CCK8,cell migration experiment,and cell formation experiment.The intracellular expression levels of mi RNA7,KLF4,VEGFA,b FGF were detected by q RT-PCR,and WB was used to detect the protein expression levels of KLF4,VEGFA,b FGF.Results1.By screening the results of high-throughput sequencing and conducting bioinformatics analysis,it was determined that KLF4 was the m RNA that was significantly differentially expressed during the DO process and was associated with angiogenic function.And mi RNA7 may regulate the expression of KLF4.The expression trend of KLF4 and mi RNA7 in tissues verified by q RT-PCR was consistent with that of sequencing results.Luciferase experiments verified the targeting relationship between mi RNA7 and KLF4.2.Results of isolation,culture and identification of EPCs in vitro: On the 3rd day after primary culture of EPCs,adherent cells appeared,which were short fusiform.On the 5th day,cell colonies began to form,and on the 7th day,cell colonies began to form.On the 10 th day,the cells showed a typical "paving stone" shape.The results of double fluorescence labeling showed that the canine EPCs expressed DL-AC-LDL in the intracellular lysosome and FITC-UEA-1 in the membrane which showed red and green respectively and the combination showed yellow.The results of immunofluorescence staining showed that the primary cultured EPCs were positive for CD34,CD133 and VEGFR2.The results of migration experiment showed that cell migration could be observed after 24 h,and the migration increased significantly after 48 h.The results of tube forming experiment showed that after 6 hours,EPCs could be observed to connect end to end in the matrix adhesive,forming tubular structure.3.After transfection of over-expressing mi RNA7 lentivirus,the proliferation,migration and tube-forming ability of EPCs decreased compared with the control group,while the expression of KLF4,VEGFA and b FGF decreased After transfection with silenced mi RNA7 lentivirus,the proliferation,migration and tube-forming ability of EPCs were increased compared with the control group,and the expressions of KLF4,VEGFA and b FGF were increased.4.Bioinformatics software mi Randa predicted that lnc RNA193740 might bind to mi RNA7,and q RT-PCR results showed that the expression trend of lnc RNA193740 was consistent with the sequencing results in the tissues.5.After transfection with over-expressing lnc RNA193740 lentivirus,the proliferation,migration,and tube-forming ability of EPCs increased compared with the control group,while the expression of mi RNA7 decreased,and the expression of KLF4,VEGFA,b FGF increased.Conclusion1.KLF4 and mi RNA7 may be involved in regulating the function of EPCs during distraction osteogenesis.2.mi RNA7 can target KLF4 to regulate the neovascularization function of endothelial progenitor cells3.lnc RNA193740 can be involved in the regulation of KLF4,thereby promoting the neovascularization ability of EPCs. |
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